Display omitted •UHPLC-MS/MS procedures for measurement of unbound and total bictegravir concentrations in different body fluids were developed.•Measurement procedures were validated using the EMA and CLSI validation guidelines.•Verification of the applicability of the procedures were performed using HIV-1 subject samples.•The measurement procedures can be useful for bictegravir pharmacokinetic/pharmacodynamic studies in plasma and cerebrospinal fluid. Bictegravir is a novel integrase strand transfer inhibitor, administrated in co-formulation with tenofovir alafenamide and emtricitabine (Biktarvy®), indicated in the management of HIV-1 infection in patients not previously treated with antiretroviral therapy. Bictegravir is highly bound to plasma proteins, and this significantly determines its clearance, solubility, and activity. These characteristics are crucial determinants of bictegravir penetration into human body compartments, as the central nervous system. We developed and validated UHPLC-MS/MS procedures to measure total and unbound bictegravir concentrations in plasma and cerebrospinal fluid. Simple protein precipitation with acetonitrile was implemented to prepare plasma and cerebrospinal fluid samples. Sample preparation was preceded by ultrafiltration for measuring unbound bictegravir concentrations. Chromatographic separations were achieved on an Acquity® UHPLC® BEHTM (2.1 × 100 mm id, 1.7 μm) reverse-phase C18 column using an isocratic mobile phase 20:80 (v/v) water/acetonitrile with 0.1% formic. Bictegravir and its internal standard (bictegravir-15N d2) were detected by electrospray ionization mass spectrometry in positive and multiple reaction monitoring modes, using transitions of 450.2→289.2/145.4 and 453.2→289.2, respectively. Ultrafiltration procedures presented non-specific bindings of (8.6 ± 1.2) % for bictegravir in plasma and (26.6 ± 3.1) % for bictegravir in cerebrospinal fluid. Linearity was observed between (10.70–8560) μg/L, (1.07–856.0) μg/L for total and unbound bictegravir in plasma, and 0.107―26.75 μg/L for total and unbound bictegravir in cerebrospinal fluid. Imprecisions, absolute relative biases, normalized-matrix factors, and normalized-recoveries were ≤14.4%, ≤13.8%, (97.4–102.5) %, and (99.8–105.1) %, respectively. No significant interferences and carry-over were observed. The validated UHPLC-MS/MS procedures could be useful for pharmacokinetic and pharmacodynamic studies.